Coenzyme A (CoA) is the major acyl group carrier in biology and an essential cofactor in intermediary metabolism. Free CoA (unacylated) and its thioesters are allosteric ligands that govern the carbon flux through key metabolic enzymes and, in turn, control energy generation. There is ample evidence that intracellular CoA levels fluctuate in response to hormones, diet, and drugs, and are dysregulated in metabolic disorders such as diabetes. CoA is produced by a universal series of reactions starting from the vitamin pantothenate, and the flux through this pathway is controlled by the first enzyme, pantothenate kinase (PanK). Mammals have multiple PanK enzymes, each with unique regulatory properties, expression patterns and intracellular distributions. The critical importance of PanK in metabolism has been extensively validated in prokaryotic systems but there are significant gaps in our understanding of the biochemical regulation and role of the multiple mammalian PanK enzymes in the physiological control of metabolism. The importance of these enzymes was brought into sharp focus in mammalian systems by the discovery of the association of mutations in the human PANK2 gene with a progressive neurological disorder called PKAN. The human PanK2 isozyme is associated with mitochondria. The initial idea was that all disease- causing mutations inactivated PanK2 enzymatic function; however, our comprehensive analysis illustrates that a significant percentage of the mutations do not inactivate PanK2 suggesting that these missense mutations alter an important regulatory feature of the protein. We propose that mitochondrial PanK has a unique regulatory role in the metabolic adaptation of CoA levels to the utilization of fatty acids as a fuel source. Specifically, CoA levels must increase for mitochondria to efficiently carry out fatty acid p-oxidation, and PanK2 senses the status of mitochondrial p-oxidation and adjusts the rate of CoA biosynthesis accordingly. The results from the last grant period reveal the novel regulatory biochemical mechanism for PanK2 activity and we will investigate the mechanistic basis for the complex control of PanK2 activity and determine if the disease-causing PanK2 mutations are altered in this property. We will also address the role of PanK2 in regulating the cellular response to metabolic shift from glucose to fatty acids in cells. These results will provide essential information about the control systems that regulate energy metabolism in mammals and establish the scientific foundation for future development of hypotheses regarding the metabolic basis for the human PKAN disease. The research requires expertise in the areas of CoA biosynthesis, lipid metabolism and protein biochemistry which are the clear strengths of the investigative team assembled to pursue this problem. [unreadable] [unreadable] [unreadable]